Most people do not suffer from autoimmunity despite the production of CD4+ T cells expressing T cell receptors (TCR) specific for self peptide (p):major histocompatibility complex 1 (MHCII) ligands. Many studies in TCR transgenic mouse models have shown that this is the case because these CD4+ T cells are deleted in the thymus or differentiate into anergic or suppressive regulatory T (Treg) cells in secondary lymphoid organs. Nevertheless, consensus on the relative contributions of these mechanisms to tolerance to all self antigens has not been reached. Fundamental questions therefore remain to be answered such as how efficient is thymic deletion, do anergic T cells exist, is the Treg cell repertoire really enriched for self pMHCIl-specific cells, and which of these mechanisms fails during autoimmunity? We will answer these questions by studying polyclonal endogenous CD4+ T cells specific for self p:MHCII ligands using a sensitive p:MHCII tetramer-based cell enrichment method. In mice, we will determine whether T cells expressing TCRs with the highest affinities for ubiquitous self p:MHCII ligands are deleted, and whether some T cells specific for p:MHCII ligands derived from peripheral tissue-specific proteins expressed in the thymus under the control ofthe Autoimmune Regulator (AIRE) escape deletion but become anergic or differentiate into Treg cells in the secondary lymphoid organs. We will attempt to confirm these hypotheses in humans by direct ex vivo tracking of the number, function, and phenotype of insulin or glutamic acid decarboxylase p:MHCII-specific CD4+ T cells from normoglycemic or type 1 diabetic people. If successful, we will have learned how efficient thymic clonal deletion is, whether anergy exists as a tolerance mechanism, and if self-reactive T cell populations are enriched for Treg cells,, all within normal polyclonal repertoires. These experiments could set the stage for future clinical trials to determine if self p:MHCII tetramer-based cell enrichment can be used as a tool for early diagnosis of diabetes or to monitor the efficacy of immunotherapy.